The present invention relates to the field of plant molecular biology, more specifically the invention relates to composition and assays for detecting the presence of specific genetic constructs in plant transformation events in a DNA sample from a cotton plant or other plant species.
Cotton is an important fiber crop in many areas of the world. The methods of biotechnology have been applied to cotton for improvement of the agronomic traits and the quality of the product. The method of introducing transgenes into cotton plants is demonstrated in U.S. Pat. No. 5,004,863. One such introduced DNA sequence important in cotton production is the cauliflower mosaic virus 35S promoter (CaMV35 or 35S) sequence. The promoter from the cauliflower mosaic virus is one of the most abundantly used genetic elements in genetically transformed crops. An important gene is the neomycin phosphotransferase sequence (nptII). The nptII sequence was originally isolated from the bacterial transposon 5. The nptII sequence has been used in many genetically transformed crops as a marker sequence. These DNA sequences have been introduced into cotton plants and are found in commercially available transformed varieties of cotton that are under production.
The expression of foreign gene sequences in plants is known to be influenced by their chromosomal position, perhaps due to chromatin structure (e.g., heterochromatin) or the proximity of transcriptional regulation elements (e.g., enhancers) close to the integration site (Weising et al., Ann. Rev. Genet 22:421-477, 1988). For a number of reasons, it is often desirable to screen for specific DNA sequences in order to confirm the presence of an event of interest. For example, it has been observed in plants and in other organisms that there may be a wide variation in levels of expression of an introduced genes among events. There may also be differences in spatial or temporal patterns of expression, for example, differences in the relative expression of a transgene in various plant tissues, that may not correspond to the patterns expected from transcriptional regulatory elements present in the introduced DNA construct(s). There is also an increasing interest in sampling lots of plant-based material for identity preservation, that is, to detect whether the material being tested contains any, all or no genetically-transformed plant material and/or the source of any transformed plant material identified in a given lot that is being tested or sampled. For this and other reasons, it is often desirable to screen or assay for a specific genetic transformation event.
One example of when it would be advantageous to be able to detect the presence of a particular event is in order to determine whether progeny of a sexual cross contain a transgene of interest. In addition, a method for detecting a particular event would be helpful for complying with various regulatory requirements, for example regulations requiring the premarket approval or labeling of foods derived from recombinant crop plants.
It is possible to detect the presence of a genetic transformation event by any well known nucleic acid detection method including DNA amplification methods, such as the polymerase chain reaction (PCR) or DNA hybridization using nucleic acid probes. These detection methods generally focus on frequently used genetic elements, such as promoters, terminators, marker genes, etc. As a result, such methods may not be useful for discriminating between different genetic transformation events that possess more than one element, particularly those produced using the same DNA construct unless additional information is known. An event-specific PCR assay is discussed, for example, by Windels et al. (Med. Fac. Landbouww, Univ. Gent 64/5b:459-462, 1999), who identified glyphosate tolerant soybean event 40-3-2 by PCR using a primer set spanning the junction between the insert and flanking DNA, specifically one primer that included sequence from the insert and a second primer that included sequence from flanking DNA.
However, these currently available methods of detecting genetic transformation events in plant material involve the use of assays that are not specific or robust enough to differentiate among specific genetic transformation events. Commonly such previously available methods involve performing multiple detection assays, each assay specific for just one genetic construct or sequence, in order to detect the presence of transgenic DNA. For example, one assay for detection of the 35S promoter that is currently in use for detection of transgenic plant material is designed to detect the presence of the 35S promoter in a sample. The problem with this is that a sample can be positive for the 35S promoter in a detection assay if it contains any one of the commercial cotton genetic transformation events, or if the sample contains another of the at least twenty-two products that currently use the 35S promoter sequence, or if the sample has been contaminated with a virus that has a similar sequence to the 35S promoter (a false positive result). Such a 35S promoter detection assay is considered to be not specific enough to say with certainty the source identity of a positive result. An assay for the 35S promoter alone cannot definitively determine whether a sample that gives a positive result does contain, for example, a sample from a transformed cotton plant or represents some other transgenic sample or represents a plant infected by a virus with a similar sequence. To detect even the current commercial cotton genetic transformation events, at least three separate event-specific PCR assays would need to be performed. More specific PCR assays (with lower false positive levels) to detect the current commercial cotton transgenic events are still needed.
A single assay to detect the presence of specific genetic transformation events in a sample lot of plant material such as cotton seed, the primers for such an assay and methods of using the assay are provided. DNA sequences are provided that comprise unique and advantageous primers that produce unique and unexpected target amplicons that span a junction sequence of an event comprising the 35S promoter and nptII coding sequences; the primers are identified as SEQ ID NO:1 and SEQ ID NO:2. Sequences highly homologous to these primers are also useful in the assay, as are the complements to each, as one would expect.
The invention accomplishes the designing of a single generic assay that detects all three of the current commercial cotton transgenic events and is more specific than current promoter or gene-specific assays. The assay methods also detect the presence of a DNA construct comprising the 35S promoter and nptII sequence in close proximity and in the same orientation in other species of plants.
The assay of the present invention uses one primer anchored in the 35S promoter and one primer anchored in the nptII sequence. The resulting amplicon of these primers span the junction region between the 35S promoter and the nptII sequence in the constructs. Different constructs of these elements will produce different amplicons. The constructs used in making the current cotton products were different and the present assay can differentiate between the two constructs used.
It is highly unlikely that any potential contaminants in a seed or other plant-material-containing sample other than from a transgenic source could produce a positive result using this assay since the 35S promoter sequence and nptII sequence are not found together in non-transgenic organisms. It was surprisingly discovered that although many primers can be generated to anchor in each element of the sequence, such primers usually are not specific and robust in their performance and produce both false positive results and/or false negative results to when used in an assay.
The primers of the present invention overcome this problem. The amplicons produced using these primers are specifically and robustly diagnostic for the nptII/35S construct used to make genetic transformation events. The amplicons produced by DNA primers homologous or complementary to SEQ ID NO:1 and SEQ ID NO:2 are an aspect of the invention.
According to another aspect of the invention, methods of detecting the presence of DNA corresponding to the nptII/35S DNA sequence in transgenic cotton or other plants, in a sample are provided. Such methods comprise: (a) contacting the sample DNA with a primer set comprising SEQ ID NO: 1 and SEQ ID NO:2 in a nucleic acid amplification reaction; (b) performing a nucleic acid amplification reaction, and (c) detecting the presence or absence of the amplicon.
These and other features, aspects, and advantages of the present invention will become better understood with regard to the following description and appended claims .
The following detailed description is provided to aid those skilled in the art in practicing the present invention. Even so, this detailed description should not be construed to unduly limit the present invention as modifications and variations in the embodiments discussed herein can be made by those of ordinary skill in the art without departing from the spirit or scope of the present inventive discovery.
All publications, patents, patent applications and other references cited in this application are herein incorporated by reference in their entirety as if each individual publication, patent, patent application or other reference were specifically and individually indicated to be incorporated by reference.
The following definitions and methods are provided to better define the present invention and to guide those of ordinary skill in the art in the practice of the present invention. Unless otherwise noted, terms are to be understood according to conventional usage by those of ordinary skill in the relevant art. Definitions of common terms in molecular biology may also be found in Rieger et al., Glossary of Genetics: Classical and Molecular, 5th edition, Springer-Verlag: New York, 1991; and Lewin, Genes V, Oxford University Press: New York, 1994. The nomenclature for DNA bases as set forth at 37 CFR xc2xa7 1.822 is used.
As used herein, the term xe2x80x9ccottonxe2x80x9d means Gossypium hirsutum and includes all plant varieties that can be bred with cotton, including wild cotton species.
As used herein, the term xe2x80x9ccomprisingxe2x80x9d means xe2x80x9cincluding but not limited to.xe2x80x9d
A transgenic xe2x80x9ceventxe2x80x9d occurs with each independent transformation of plant cells with heterologous DNA, i.e., a nucleic acid construct that includes a transgene of interest; regeneration of a population of plants resulting from the insertion of the transgene into the genome of the plant; and selection of a particular-plant characterized by insertion into a particular genome location. The term xe2x80x9ceventxe2x80x9d also refers to DNA from the original transformant comprising the inserted DNA and flanking genomic sequence immediately adjacent to the inserted DNA that would be expected to be transferred to a progeny that receives inserted DNA including the transgene of interest as the result of a sexual cross of one parental line that includes the inserted DNA (e.g., the original transformant and progeny resulting from selfing) with a parental line that does not contain the inserted DNA.
A xe2x80x9cprobexe2x80x9d is an isolated nucleic acid to which is desirably attached a conventional detectable label or reporter moiety, e.g., a radioactive isotope, ligand, chemiluminescent agent, vitamin, steroid or enzyme. Such a probe is complementary to a strand of a target nucleic acid, in the case of the present invention, to a strand of genomic DNA from transformation event nptII/35S, whether from a cotton plant, other plant containing the event, or from a sample that includes DNA from the event. Probes according to the present invention include not only deoxyribonucleic or ribonucleic acids but also polyamides and other probe materials that bind specifically to the target event DNA sequence and can be used to detect the presence of that target DNA sequence.
xe2x80x9cPrimersxe2x80x9d are isolated nucleic acids that can be annealed to a complementary target DNA strand by nucleic acid hybridization to form a hybrid between the primer and the target DNA strand, then extended along the target DNA strand by a polymerase, e.g., a DNA polymerase. Primer pairs of the present invention refer to their use for amplification of a target nucleic acid sequence, e.g., by the polymerase chain reaction (PCR) or other conventional nucleic-acid amplification methods.
Probes and primers are generally 11 nucleotides or more in length, preferably 18 nucleotides or more, more preferably 21 nucleotides or more. Such probes and primers hybridize specifically to a target sequence under high stringency hybridization conditions. Preferably, probes and primers according to the present invention have complete sequence similarity with the target sequence, although probes differing from the target sequence and that retain the ability to hybridize to target sequences may be designed by conventional methods.
Methods for preparing and using probes and primers are described, for example, in Molecular Cloning: A Laboratory Manual, 2nd ed., vol. 1-3, ed. Sambrook el al., Cold Spring Harbor Laboratory Press, Cold Spring Harbor, N.Y., 1989 (hereinafter, xe2x80x9cSambrook et al., 1989xe2x80x9d); Current Protocols in Molecular Biology, ed. Ausubel et al., Greene Publishing and Wiley-Interscience, New York, 1992 (with periodic updates) (hereinafter, xe2x80x9cAusubel et al., 1992xe2x80x9d); and Innis et al., PCR Protocols: A Guide to Methods and Applications, Academic Press: San Diego, 1990. PCR-primer pairs can be derived from a known sequence, for example, by using computer programs intended for that purpose such as Primer (Version 0.5, (copyright) 1991, Whitehead Institute for Biomedical Research, Cambridge, Mass.).
The nucleic acid probes and primers of the present invention hybridize under stringent conditions to the nptII/35S target DNA sequence. Any conventional nucleic acid hybridization or amplification method can be used with the primers of the present invention to identify the presence or absence of DNA from the target, specific genetic transformation event in a sample.
Nucleic acid molecules or fragments thereof are capable of specifically hybridizing to other nucleic acid molecules under certain circumstances. As used herein, two nucleic acid molecules are said to be capable of specifically hybridizing to one another if the two molecules are capable of forming an anti-parallel, double-stranded nucleic acid structure. A nucleic acid molecule is said to be the xe2x80x9ccomplementxe2x80x9d of another nucleic acid molecule if they exhibit complete complementarity. As used herein, molecules are said to exhibit xe2x80x9ccomplete complementarityxe2x80x9d when every nucleotide of one of the molecules is complementary to a nucleotide of the other. Two molecules are said to be xe2x80x9cminimally complementaryxe2x80x9d if they can hybridize to one another with sufficient stability to permit them to remain annealed to one another under at least conventional xe2x80x9clow-stringencyxe2x80x9d conditions. Similarly, the molecules are said to be xe2x80x9ccomplementaryxe2x80x9d if they can hybridize to one another with sufficient stability to permit them to remain annealed to one another under conventional xe2x80x9chigh-stringencyxe2x80x9d conditions. Conventional stringency conditions are described by Sambrook et al., 1989, and by Haymes et al, In: Nucleic Acid Hybridization, A Practical Approach, IRL Press, Washington, D.C. (1985). Departures from complete complementarity are therefore permissible, as long as such departures do not completely preclude the capacity of the molecules to form a double-stranded structure. In order for a nucleic acid molecule to serve as a primer or probe it need only be sufficiently complementary in sequence to be able to form a stable double-stranded structure under the particular solvent and salt concentrations employed.
As used herein, a substantially homologous sequence is a nucleic acid sequence that will specifically hybridize to the complement of the nucleic acid sequence to which it is being compared under high stringency conditions. Appropriate stringency conditions which promote DNA hybridization, for example, 6.0xc3x97sodium chloride/sodium citrate (SSC) at about 45xc2x0 C., followed by a wash of 2.0xc3x97SSC at 50xc2x0 C., are known to those skilled in the art or can be found in Current Protocols in Molecular Biology, John Wiley and Sons, N.Y. (1989), 6.3.1-6.3.6. For example, the salt concentration in the wash step can be selected from a low stringency of about 2.0xc3x97SSC at 50xc2x0 C. to a high stringency of about 0.2xc3x97SSC at 50xc2x0 C. In addition, the temperature in the wash step can be increased from low stringency conditions at room temperature, about 22xc2x0 C., to high stringency conditions at about 65xc2x0 C. Both temperature and salt may be varied, or either the temperature or the salt concentration may be held constant while the other variable is changed.
In one exemplary embodiment, the nucleic acid primers of the present invention will specifically hybridize to a target portion of the nptII/35S event under moderately stringent conditions, for example at about 2.0xc3x97SSC and about 65xc2x0 C. In another exemplary embodiment, the nucleic acid primers of the present invention will specifically hybridize to a target portion of the nptII/35S event under high stringency conditions.
Regarding the amplification of the target portion nucleic acid sequence (e.g., by PCR) using the primers of the present invention, xe2x80x9cstringent conditionsxe2x80x9d are conditions that permit the primer pair to hybridize only to the target nucleic-acid sequence to which the primer pair comprising SEQ ID NO: 1 and SEQ ID NO: 2, would bind and preferably to produce the unique amplification product, the amplicon, in a DNA amplification reaction.
The term xe2x80x9cspecific for (a target sequence)xe2x80x9d indicates that a probe or primer hybridizes under stringent hybridization conditions only to the target sequence in a sample comprising the target sequence.
As used herein, xe2x80x9camplified DNAxe2x80x9d or xe2x80x9campliconxe2x80x9d refers to the product of nucleic-acid amplification of a target nucleic acid sequence that is part of a nucleic acid template. The use of the term xe2x80x9campliconxe2x80x9d specifically excludes primer dimers that may be formed in the DNA thermal amplification reaction.
Nucleic-acid amplification can be accomplished by any of the various nucleic-acid amplification methods known in the art, including but not limited to the polymerase chain reaction (PCR), LCR, TAS, 3SR, NASBA RCA and Q.beta. amplification.
A variety of amplification methods are known in the art and are described, inter alia, in U.S. Pat. Nos. 4,683,195 and 4,683,202 and in PCR Protocols: A Guide to Methods and Applications, ed. Innis et al., Academic Press, San Diego, 1990 and PCR Applications: Protocols For Functional Genomics, ed. Innis et al., Academic Press, San Diego, 1999. These methods as well as other methods known in the art of DNA amplification may be used in the practice of the present invention. The sequence of the specific transformation event of interest, the nptII/35S sequence, in cotton or other species of plants, can be verified by use of the primers comprising SEQ ID NO: 1 and SEQ ID NO: 2 of the invention provided herein in any suitable DNA amplification assay.
The readout in the assay may be fluorescent or ELISA-based. A signal indicates presence of the nptII/35S DNA sequence from a transgenic event, due to successful amplification, hybridization, and evaluation.
Taqman(copyright) (PE Applied Biosystems, Foster City, Calif.) is described as a method of detecting and quantifing the presence of a DNA sequence and is fully understood in the instructions provided by the manufacturer. Briefly, a FRET oligonucleotide probe is designed which overlaps the genomic flanking and insert DNA junction. The FRET probe and PCR primers (one primer in the insert DNA sequence and one in the flanking genomic sequence) are cycled in the presence of a thermostable polymerase and dNTPs. Hybridization of the FRET probe results in cleavage and release of the fluorescent moiety away from the quenching moiety on the FRET probe. A fluorescent signal indicates the presence of the flanking/transgene insert sequence due to successful amplification and hybridization.
DNA detection kits can be developed using the compositions disclosed herein and the methods well known in the art of DNA detection. The kits are useful for identification of a transgenic event, containing a nptII/35S DNA construct, in a sample. The kits contain DNA sequences homologous or complementary to SEQ ID NO:1 and to SEQ ID NO:2. These DNA sequences are used as primers in DNA amplification reactions, or as probes in a DNA hybridization method.
The transgene genetic elements referred to herein are the cauliflower mosaic virus 35S promoter (P-CaMV.35S (Odell, et al. Nature 313:810-812, 1985), operably connected to the neomycin phosphotransferase gene (NPTII) (Fraley et al. Proc Natl. Acad Sci USA 80:4803-4807, 1983).
The assay of the present invention preferably includes an internal control step comprising including primers derived from native cotton DNA. Production of an amplicon of known size from these primers is a check for proper functioning of the assay, e.g., the use of proper reagents and conditions. One set of such primers derived from native cotton DNA are SEQ ID NO: 5 and SEQ ID NO: 6. They produce an amplicon of approximately 600 base pairs which is easily separable from an amplicon of the transgenic DNA, which in the current cotton products would be either about 340 base pairs or 428 base pairs as discussed below.